A rapid, sensitive, dot-blot assay for insulin-like growth factor-II (IGF-II) based on chemiluminescence methodology has been further evaluated for possible interference by IGF binding proteins (IGFBPs). Six different IGFBPs were tested in the dot blot assay at concentrations up to 60-fold the concentration of IGF-II. The IGF-II signal was not diminished. By contrast, in a conventional radioimmunoassay using the same monoclonal antibody to IGF-II, the binding proteins produced significant interference, causing 23% to 87% competition of binding of 125I-IGF-II to the antibody. Thus the IGF-II dot blot assay is unique in allowing direct measurement of IGF-II in conditioned medium from cells in culture without interference by IGFBPs. Addition of IGF-I to MG-63 human osteosarcoma cells resulted in rapid activation of extracellular signal regulated kinase 2 (ERK2) as assessed by ion exchange HPLC and ERK kinase antibodies. ERK1 appeared to be already activated in serum-starved MG-63 cells and not to be further activated following addition of IGF-I. Thus ERK2 activation may be part of the signaling pathway for the IGF-I receptor. Further evaluation of fibroblasts from two patients with deletion of the distal long arm of chromosome 15 showed evidence for decreased expression of IGF-I receptor mRNA in one of the patients compared to age-matched controls. Evaluation of receptor function in a bioassay which measured the incorporation of [3H]thymidine into DNA in the presence of a full range of IGF-I concentrations showed no difference in the ED50 for IGF-I between the patient and control fibroblasts. We conclude from these findings and our earlier results that although there is evidence for decreased IGF-I receptor expression in fibroblasts from patients with chromosome 15q deletion syndrome, receptor function as assessed by cellular response to IGF-I is not impaired.